Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor

Abstract

Tissue factor (TF) plays an important role in hemostasis, inflammation, angiogenesis, and the pathophysiology of atherosclerosis and cancer. In this article we uncover a mechanism in which protein S, which is well known as the cofactor of activated protein C, specifically inhibits TF activity by promoting the interaction between full-length TF pathway inhibitor (TFPI) and factor Xa (FXa). The stimulatory effect of protein S on FXa inhibition by TFPI is caused by a 10-fold reduction of the K(i) of the FXa/TFPI complex, which decreased from 4.4 nM in the absence of protein S to 0.5 nM in the presence of protein S. This decrease in K(i) not only results in an acceleration of the feedback inhibition of the TF-mediated coagulation pathway, but it also brings the TFPI concentration necessary for effective FXa inhibition well within range of the concentration of TFPI in plasma. This mechanism changes the concept of regulation of TF-induced thrombin formation in plasma and demonstrates that protein S and TFPI act in concert in the inhibition of TF activity. Our data suggest that protein S deficiency not only increases the risk of thrombosis by impairing the protein C system but also by reducing the ability of TFPI to down-regulate the extrinsic coagulation pathway.

Fig. 1.

Thrombin generation in plasma. (A) Thrombin generation was initiated in plasma (in the presence of APC-inhibiting antibodies) with 1.4 pM TF, 10 μM phospholipid vesicles, and 16 mM CaCl₂ (final concentrations) and followed continuously with the fluorogenic substrate I-1140 (Z-Gly-Gly-Arg-7-amino-4-methylcoumarin·HCl). •, normal plasma with protein S; ○, normal plasma without protein S; ▴, TFPI-depleted plasma with protein S; ▵, TFPI-depleted plasma without protein S. A typical experiment is shown. (B) ETP values of TFPI-depleted plasma reconstituted with varying amounts of full-length TFPI (circles) or TFPI_1–161 (triangles) in the presence of protein S (closed symbols) or absence of protein S (open symbols). The averages of two independent experiments are shown.

Fig. 2.

Inhibition of TF/FVIIa-catalyzed FX activation by full-length TFPI and protein S. Activation of 160 nM FX by 1 pM TF/FVIIa was followed in reaction mixtures that contained 15 μM phospholipids, 3 mM Ca²⁺, no TFPI and no protein S (♦), 100 nM protein S (■), 1 nM TFPI (•), and 1 nM TFPI and 100 nM protein S (▴). Averages of three independent measurements ± SD are shown.

Fig. 3.

Influence of protein S on FXa inhibition by full-length TFPI. (A) Conversion of 0.5 mM S2222 by 0.2 nM FXa was monitored in reaction mixtures containing 10 μM phospholipids, 3 mM CaCl₂, and either no protein S (dashed line) or 160 nM protein S (dotted line). Without TFPI, S2222 conversion by FXa was linear in time (with or without protein S present, solid line). At the time indicated 1.54 nM TFPI was added. The absorbance data were fitted to Eq. 2. (B) First derivatives of the fitted curves representing the change in free FXa with time. A typical experiment is shown.

Fig. 4.

Effect of protein S on v_o and v_s calculated from time courses of FXa inhibition by TFPI. Progress curves of S2222 conversion by FXa were measured at varying concentrations of TFPI in the absence and presence of protein S. Fitting of the progress curves to Eq. 2 yielded values for v_o (A) and v_s (B) as function of the TFPI concentration. Final concentrations were 0.2 nM FXa, 10 μM phospholipid vesicles (20:60:20 DOPS/DOPC/DOPE), 3 mM CaCl₂, 0.5 mM S2222, 0–3.9 nM TFPI, and no protein S (○) or 100 nM protein S (•). The average of two independent experiments is shown.